Abstract The emergence of multi-drug resistant Gram-negative bacteria creates a compelling need for the development of new effective antibiotics. Efforts to discover new chemotypes for the treatment of Gram-negative bacteria are, however, often confounded by the organism?s natural defenses. These treatment barriers include such diverse elements as reduced membrane permeability, widespread efflux activity and plasmid-encoded resistance genes. Inspired by the natural product ?-thujaplicin, we have established a program to develop therapeutic agents based on the tropolone core, a relatively unexplored metal-directing pharmacophore. Through direct methods a putative target for these compounds, the essential metalloenzyme Enolase, was identified in our laboratory. Functionally, enolase is a lyase and a key player in glycolysis where it is responsible for the reversible generation of phosphoenolpyruvate (PEP). PEP, a key intermediate in metabolism/energy production, also functions as an integral building block in bacterial cell wall biosynthesis. In addition, enolase plays an important but less well-defined role as a component of the RNA degradasome, which is responsible for the digestion of ribonucleic acids. Unlike typical natural products, members of the tropolone family are relatively small, low molecular weight compounds with low logD values. As such, they are more reminiscent of early lead compounds than they are of complex natural extracts. These physicochemical attributes in conjunction with the presence of multiple sites available for synthetic diversification suggest that the tropolones can benefit from the introduction of additional potency-increasing functionality without compromising the drug-like properties required for Gram-negative activity. Herein, two specific aims are proposed. The first aim focuses on the synthesis and evaluation-driven optimization of the tropolone leads against both the biochemical target, enolase, as well as four important Gram-negative pathogens. In addition to generating enzymatic profiles for the compounds cell-based selectivities, physicochemical properties and metabolic processing of the compounds will also be evaluated. In a second, independent aim the major pharmacological effects of the compounds, both at the enzyme and cell level, will be investigated. Conditions for the acquisition of high-resolution co-crystal structures of tropolone leads with both the bacterial and human isoforms will be optimized. Newly generated co-crystal structures will be employed to inform existing structure-activity relationships and enhance efforts in Aim 1 to optimize potency and selectivity. In a parallel effort, several ?tool compounds? will be used, to enhance the overall understanding of the pharmacological potential of targeting the enolase enzyme.